The SCF-Fbw7 ubiquitin ligase targets proteins for destruction by the ubiquitin-proteasome system. Fbw7 is the SCF component that recognizes substrates and brings them into proximity with the ubiquitylation machinery. Fbw7's substrates include proteins with key roles in cell division, growth, and differentiation, and these include broadly acting oncoproteins (Myc, cyclin E, Notch, and Jun). Accordingly, Fbw7 is a tumor suppressor that is commonly mutated in cancers. The research proposed in this application seeks to develop a comprehensive and mechanistic framework for understanding Fbw7 function and how Fbw7 mutations lead to cancer. These studies are needed to understand its mechanisms of tumor suppression and to develop therapy targeting the Fbw7 pathway. Fbw7 binds to substrates via conserved phosphodegron motifs (CPDs). These complex interactions are regulated both by the phosphodegrons themselves and by Fbw7 dimerization. The goals of Aim 1 are to study how Fbw7 dimers and CPDs regulate substrate ubiquitylation, and to ascertain the physiologic functions of Fbw7 dimers. We will accomplish these goals by using in vitro approaches to study Fbw7 binding and substrate ubiquitylation, and by using gene targeting in human cells and mice to study endogenous Fbw7 dimer function. Importantly, the SCF-Fbw7 shares structural and functional features with other cullin-ring ligases (CRLs) implicated in human diseases, including dimerization. The impact of these studies may thus extend beyond the Fbw7 pathway and reveal common mechanisms of CRL function. Fbw7 mutations occur commonly in cancers and often involve Fbw7 arginine residues that contact substrate CPDs. Because Fbw7 targets multiple oncoproteins, its tumor suppression functions are complex and the mechanisms by which Fbw7 mutations promote cancer are poorly understood. However, an in-depth understanding of the molecular pathogenesis of Fbw7 mutations is needed in order to develop therapy targeting the Fbw7 pathway. The goals of this aim are to develop models of Fbw7-associated cancer and to understand how Fbw7 mutations contribute to carcinogenesis. We will approach these goals by using gene targeting in mice and human cells to mimic tumor-derived Fbw7 mutations, and by performing a functional genomic screen in mice to identify genes that cooperate with Fbw7 loss during tumorigenesis. These models will be used to gain a mechanistic understanding of Fbw7 mutations, and they will provide key platforms for future studies of targeted therapy.